Structure-Activity Relationship (SAR) study provides valuable insights for understanding intermolecular interactions between a protein or peptide and other biologically active molecules. In their natural environment, peptides or proteins adopt unique, conformationally-constrained structures in order to recognize and bind to their binding partners, and to form a molecular complex therewith, which in turn elicit particular activities. Examples of protein-protein binding partners include enzyme-substrate, ligand-receptor, and antigen-antibody. Determination of the conformation of a peptide in its native form, therefore, become crucial for closely mimicking its in vivo activity and rationally designing its analogues which may be useful as drugs.
Most small peptides are highly flexible and do not typically adopt unique solution conformations; in particular, they do not maintain the structure that the same sequence adopts in the native protein. The lack of fixed structure reduces the affinity the peptide might have for a target (for entropic reasons) and makes determination of the active conformation of the molecule extremely difficult. Because of this, many strategies have been described to introduce constraints into peptides (such as D-amino acids, disulfide or other crosslinks), or to replace parts of the peptide with more rigid non-peptide scaffolds. Indeed, such peptidomimetics have been widely used to perform structure-activity studies in a systematic way to provide information about the specific amino acid residues or functional groups in a peptide that are adaptable to a particular conformation and are important to biological activities.
Several constrained protein scaffolds, capable of presenting a protein of interest as a conformationally-restricted domain have been identified, including minibody structures (Bianchi et al. (1994) J Mol Biol 236:649-659), loops on β-sheet turns, coiled-coil stem structures (Myszka & Chaiken (1994) Biochem 33:2363-2372), zinc-finger domains, cysteine-linked (disulfide) structures, transglutaminase linked structures, cyclic peptides, helical barrels or bundles, leucine zipper motifs (Martin et al. (1994) EMBO J. 13:5303-5309), and etc. Of the identified protein scaffolds, β-turns have been implicated as an important site for molecular recognition in many biologically active peptides. Smith & Pease (1980) CRC Crit Rev Biochem 8:315-300. Consequently, peptides containing conformationally constrained β-turns are particularly desirable. The great majority of the identified β-turn bearing peptides are cyclopeptides which have been generated by the cyclization of a peptide similar to a sequence in the natural substrate. Milner-White (1989) Trends Pharmacol Sci 10:70-74. These cyclopeptides, however, may still retain significant flexibility. For this reason, many studies have attempted to introduce rigid, nonpeptide compounds which mimic the β-turn. Peptides with such nonpeptide β-turn mimic provide useful leads for drug discovery. Ball & Alewood (1990) J Mol Recog 3:55-64; WO 94/03494 (Kahn).
One of the revolutionary advances in drug discovery is the development of combinatorial libraries. Combinatorial libraries are a collection of different molecules, such as peptides, that can be made synthetically or recombinantly. Combinatorial peptide libraries contain peptides in which all amino acids have been incorporated randomly into certain or all positions of the peptide sequence. Such libraries have been generated and used in various ways to screen for peptide sequences which bind effectively to target molecules and to identify such sequences.
Many methods for generating peptide libraries have been developed and described. For example, members of the peptide library can be created by split-synthesis performed on a solid support such as polystyrene or polyacrylamide resin, as described by Lam et al. (1991) Nature 354:82 and PCT publication WO 92/00091. Another method disclosed by Geysen et al., U.S. Pat. No. 4,833,092 involves the synthesis of peptides in a methodical and predetermined fashion, so that the placement of each library member peptide gives information concerning the synthetic structure of that peptide.
Considerable effort has been devoted to introducing structural constraints into combinatorial peptide libraries so that the member peptides represent more closely to their native counterparts. Houston et al. U.S. Pat. No. 5,824,483 describes a synthetic peptide library containing peptides featuring α-helical conformation and thus capable of forming coiled-coil dimers with each other. McBride et al. (1996) J Mol Biol 259:819-827 describe a synthetic library of cyclic peptides mimicking the anti-tryptic loop region of an identified proteinase inhibitor.
A complementary method for peptide library-based lead discovery is display of libraries on filamentous bacteriophages. This method allows the preparation of libraries as large as 1010-1012 unique peptide members, many orders of magnitude larger than libraries that may be prepared synthetically. In addition to large library sizes, advantages of phage display include ease of library construction (Kunkel mutagenesis), coupling of the binding entity (displayed peptide) to a unique identifier (its DNA sequence), a selection protocol for amplifying rare binding clones in a pool, and the high fidelity of biosynthesis (compared to synthetic methods). Furthermore, rapid and inexpensive selection protocols are available for identifying those library members that bind to a target of interest. However, only natural peptides composed of L-amino acids may be displayed on phage, so the problem of defining three-dimensional structure-activity relationships is more difficult than it might be for a constrained peptidomimetic containing non-naturally occurring peptides or nonpeptide compounds. One possible solution to this problem is to use the structural constraints of a folded protein to present small variable peptide segments. Indeed, several small, stable proteins have been proposed as peptide display scaffolds. Nygren & Uhlen (1997) Curr. Opin. Struct. Biol. 7:463-469; Vita et al. (1998) Biopolymers 47:93-100; Vita et al. (1999) Proc. Natl. Acad. Sci. USA 96:13091-13096; Smith et al. (1998) J. Mol. Biol. 277:317-332; Christmann et al. (1999) Protein Engng. 12:797-806. Unfortunately, it is not clear that protein ligands obtained by this approach could be transformed to small-molecule drug leads. Epitope transfer from proteins to small peptides or to non-peptide small molecules remains an extremely challenging problem. Cochran (2000) Chem. Biol. 7:R85-R94.
Therefore, despite of extensive studies of the rules governing conformational preferences in natural peptides and the existence of several peptide library systems, those features necessary for structural stability of natural peptides remain poorly understood. In particular, there has been little systematic or quantitative assessment of the effect of residue substitutions and non-covalent interactions on structure.